The central nervous system (CNS) requires a tightly controlled environment free of toxins and pathogens to provide the proper chemical composition for neural function. This environment is maintained by the ‘blood brain barrier’ (BBB), which is composed of blood vessels whose endothelial cells display specialized tight junctions and extremely low rates of transcellular vesicular transport (transcytosis)1–3. In concert with pericytes and astrocytes, this unique brain endothelial physiological barrier seals the CNS and controls substance influx and efflux4–6. While BBB breakdown has recently been associated with initiation and perpetuation of various neurological disorders, an intact BBB is a major obstacle for drug delivery to the CNS7–10. A limited understanding of the molecular mechanisms that control BBB formation has hindered our ability to manipulate the BBB in disease and therapy. Here, we identify mechanisms governing the establishment of a functional BBB. First, using a novel embryonic tracer injection method, we demonstrate spatiotemporal developmental profiles of BBB functionality and find that the mouse BBB becomes functional at embryonic day 15.5 (E15.5). We then screen for BBB-specific genes expressed during BBB formation, and find that major facilitator super family domain containing 2a (Mfsd2a) is selectively expressed in BBB-containing blood vessels in the CNS. Genetic ablation of Mfsd2a results in a leaky BBB from embryonic periods through adulthood, while maintaining the normal patterning of vascular networks. Electron microscopy examination reveals a dramatic increase in CNS endothelial cell vesicular transcytosis in Mfsd2a−/− mice, without obvious tight junction defects. Finally we show that MFSD2A endothelial expression is regulated by pericytes to facilitate BBB integrity. These findings identify MFSD2A as a key regulator of BBB function that may act by suppressing transcytosis in CNS endothelial cells. Further our findings may aid in efforts to develop therapeutic approaches for CNS drug delivery.
Summary
The blood-brain barrier (BBB) provides a constant homeostatic brain environment that is essential for proper neural function. An unusually low rate of vesicular transport (transcytosis) has been identified as one of the two unique properties of central nervous system (CNS) endothelial cells, relative to peripheral endothelial cells, that maintain the restrictive quality of the BBB. However, it is not known how this low rate of transcytosis is achieved. Here we provide a mechanism whereby the regulation of CNS endothelial cell lipid composition inhibits specifically the caveolae-mediated transcytotic route readily used in the periphery. An unbiased lipidomic analysis reveals significant differences in endothelial cell lipid signatures from the CNS and periphery, which underlie a suppression of caveolae vesicle formation and trafficking in brain endothelial cells. Furthermore, lipids transported by Mfsd2a establish a unique lipid environment that inhibits caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensure BBB integrity.
SUMMARY
Neurovascular interactions are essential for proper brain function. While the effect of neural activity on cerebral blood flow has been extensively studied, whether neural activity influences vascular patterning remains elusive. Here, we demonstrate that neural activity promotes the formation of vascular networks in the early postnatal mouse barrel cortex. Using a combination of genetics, imaging, and computational tools to allow simultaneous analysis of neuronal and vascular components, we found that vascular density and branching were decreased in the barrel cortex when sensory input was reduced by either a complete deafferentation, a genetic impairment of neurotransmitter release at thalamocortical synapses, or a selective reduction of sensory-related neural activity by whisker plucking. In contrast, enhancement of neural activity by whisker stimulation led to an increase in vascular density and branching. The finding that neural activity is necessary and sufficient to trigger alterations of vascular networks reveals a novel feature of neurovascular interactions.
Extensive neuronal cell death during development is believed to be due to a limiting supply of neurotrophic factors. In vitro studies suggest that axon guidance molecules directly regulate neuronal survival, raising the possibility that they play a direct role in neuronal cell death in vivo. However, guidance errors may also influence survival indirectly due to loss of target-derived neurotrophic support. The role of guidance molecules in neuronal death in vivo has thus been difficult to decipher. Semaphorin3A, a repulsive guidance cue for sensory neurons, can induce sensory neuron death in vitro. Null mice studies of the Semaphorin3A coreceptors showed that guidance activity is mediated by PlexinA4, but PlexinA3 partially compensates in PlexinA4 Ϫ/Ϫ mice. Here we demonstrate that both Plexins contribute to Sema3A-induced cell death in vitro, albeit in a different hierarchy. PlexinA3 is absolutely required, while PlexinA4 makes a smaller contribution to cell death. We found that PlexinA3 Ϫ/Ϫ mice, which, unlike PlexinA4 Ϫ/Ϫ mice, do not exhibit sensory axon patterning defects, show reduced neuronal apoptosis and an increased number of DRG neurons. Semaphorin3A involvement in neuronal death in vivo was demonstrated by a sensitization experiment using the proapoptotic effector Bax. Our results identify Plexins as mediators of Semaphorin-induced cell death in vitro, and provide the first evidence implicating Semaphorin/Plexin signaling in neuronal survival independent of its role in axon guidance. The results also support the idea that naturally occurring neuronal cell death reflects not only competition for target-derived trophic factors, but also the action of proapoptotic signaling via a Semaphorin/Plexin pathway.
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